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Purification method of porcine circovirus II type virus-like particles

A porcine circovirus and type 2 virus technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, recovery/purification, etc., can solve the problem of poor purification effect, low pertinence, and low efficiency of virus-like particle purification methods And other issues

Inactive Publication Date: 2017-05-31
TIANJIN RINGPU BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The present invention aims at the technical defects of the prior art, and provides a method for purifying porcine circovirus type 2 virus-like particles, so as to solve the technical problem of low efficiency of the purification method for virus-like particles in the prior art
[0005] Another technical problem to be solved by the present invention is that the purification method for virus-like particles in the prior art has relatively cumbersome steps when reaching the required purity level
[0006] Another technical problem to be solved by the present invention is that the purification method for virus-like particles in the prior art is not very specific to virus-like particles with a specific structure, and the purification effect is not good.

Method used

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  • Purification method of porcine circovirus II type virus-like particles
  • Purification method of porcine circovirus II type virus-like particles
  • Purification method of porcine circovirus II type virus-like particles

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] The acquisition of the recombinant baculovirus of embodiment 1 artificial modification PCV2CAP gene

[0039]Under the premise of keeping the amino acid unchanged, the codons are transformed into baculovirus-biased codons to increase the expression level. When performing codon optimization, the codons most frequently used by insect cells are preferred. Using the PCV2b isolate CAP gene sequence (GenBank No.EU257511.1) as the original template, the sequence of the PCV2CAP gene was artificially synthesized according to the codon preference. The modified gene sequence is shown in SEQ ID NO2, and the gene sequence comparison before and after modification Circumstances as attached figure 1 shown. The amino acid sequence encoded by the gene before and after the above modifications is shown in SEQ ID NO3.

[0040] The artificially modified PCV2CAP gene fragment was cloned into the pUC57 vector, and a large number of target genes were recovered by double digestion with BamHI a...

Embodiment 2

[0042] The identification of embodiment 2 recombinant baculovirus PCV2

[0043] 1 PCR identification

[0044] Extract P1 generation baculovirus DNA (extraction steps are carried out according to the instructions of Tiangen virus genomic DNA / RNA extraction kit), and use primer PCV2-CAP-S / A to amplify the CAP gene by PCR to identify whether the foreign gene is integrated into the recombinant in the baculovirus genome.

[0045] 2SDS-PAGE detection of Cap protein expression

[0046] Prepare 100-200 mL of sf9 cells in the logarithmic phase with good cell viability, and the cell density is 2×106 cells / mL. Aseptically inoculate up to 0.5 mL of baculovirus into the cells, and the amplification generally takes 4 to 5 days. Harvest the virus, centrifuge at 3000rmp4°C for 15min, and aseptically harvest the rod virus and store it in the dark at 4°C. The harvested recombinant baculovirus was centrifuged, the supernatant and the precipitate were separated, the precipitate was resuspende...

Embodiment 3

[0051] The purification of embodiment 3 recombinant baculovirus PCV2

[0052] Pure at first

[0053] Centrifuge the recombinant baculovirus PCV2 venom at 9500rpm at 4°C for 30min, separate the supernatant, resuspend the pellet with PBS, freeze and thaw the pellet three times, break the cells with a homogenizer, centrifuge at 9500rpm at 4°C for 30min, and collect the supernatant. Adjust the pH of the sample to 7.5, pass the supernatant through a 0.45 μm filter, and use the filtrate as a column sample.

[0054] Two fine purification

[0055] 1. Pack the column: According to the ratio recommended in the instructions, fill 10ml of Capto Core700 filler in a column with a column height of 10cm, which is about half of the column volume.

[0056] 2 Washing with water: Wash the column with one column volume (1CV) of distilled water. The flow rate is about 1.6ml / min

[0057] 3 Equilibration: Equilibrate the column with at least 5CV of buffer or wait until the UV baseline, pH and con...

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Abstract

The invention provides a purification method of porcine circovirus II type virus-like particles. The method is a new purification method developed through experiment measures centring on the self-characteristics of the PCV2 virus-like particles and the characteristics of an expression system of the PCV2 virus-like particles. The method comprises the steps that firstly, cell lysis is achieved through comprehensive utilization of freezing-thawing and ultrasonic treatment measures; secondly, a great amount of cell debris and macromolecule impurities are removed by means of a filter plate module, and sterile supernatant liquid is obtained; thirdly, the supernatant liquid is concentrated by means of a 100kD hollow fiber membrane system, and a concentrated virus antigen solution is obtained; fourthly, chromatographic purification is conducted by means of a Sepharose 4FF molecular sieve chromatographic purification system in optimized operation conditions. The method is designed closely aiming at the characteristics of the PCV2 virus-like particles, and particularly suitable for the purification of recombinant baculovirus PCV2 virus-like particles. By means of the method, the impurities can be more efficiently removed and separated to obtain target proteins, and target antigens can be effectively recycled.

Description

technical field [0001] The invention relates to the technical field of purification of biological products, in particular to a method for purifying porcine circovirus type 2 virus-like particles. Background technique [0002] Porcine circovirus (PCV) is one of the smallest viruses known so far, and the CAP protein is the main antigen of PCV2, which can induce the production of neutralizing antibodies. At present, epidemiological surveys show that PCV2 has been widely distributed in the world, and there are almost no seronegative pigs in large-scale farms. Since the first outbreak of PMWS in Canada, PMWS infections have been diagnosed on five continents of the world, posing a great threat to most pig farming countries. [0003] Due to the importance of prevention and treatment of PCV2, the preparation of PCV2 vaccine has become one of the hot spots in the research and development of biological products. The PCV2 subunit genetic engineering vaccine expresses the PCV2 structu...

Claims

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Application Information

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IPC IPC(8): C12N7/04C12N7/02C12R1/93
Inventor 郭慧娟李亚杰杨保收付旭彬梁武
Owner TIANJIN RINGPU BIO TECH
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